PROTEIN PROFILING AND ENZYMATIC STUDIES OF PESTICIDE

DEGRADING BACTERIA

Research Proposal
By
Chinmay Rout
MSc Microbiology
Roll No- 22BS001MB2

Research adviser
Dr. Jyoti Ranjan Rout
Research committee member
Dr. Pradipta Ranjan Rauta
Dr. Dhiraj Kumar Nanda

AIPH UNIVERSITY, BHUBNESWAR

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 ❖ INTRODUCTION :
Pesticides are important agrochemicals that safeguard crops from pests in modern farming.
These substances are necessary to stop pest attacks, ensure the quality, and prevent losses in
various agricultural products(Castrejón-Godínez et al., 2022). Pesticides that contain
organophosphorus compounds, such as glyphosate, chlorpyrifos, parathion, methyl parathion,
diazinon, coumaphos, monocrotophos, fenamiphos and phorate, have been widely used since
1952 , and their effects and behavior in the environment have been researched. These pesticides
are composed of various substances, such as monocrotophos, phosphoric acid, and moncorn,
that have a similar chemical structure derived from phosphoric acid, and they pollute many
aquatic and land ecosystems . Besides harming the intended organisms, these pesticides also
damage the non-target organisms and the environment, causing the deterioration of soil
components due to the accumulation of toxic residues . These pesticides may cause cancer, and
they also affect the growth of aquatic invertebrates and trigger skin allergies and eye
inflammation in humans . Eating food that has been sprayed with these pesticides can lead to
health problems such as asthma, allergies and sensitivities, and it can also interfere with the
hormones of the reproductive system and the fetus . Some of the poisons in these pesticides
affect the sodium channels in the nerve cells, resulting in an overactive state in the central
nervous system . Pesticides are chemicals that are made by industrial methods. The FAO said
in 2001 that more than 700 kinds of pests could not be killed by pesticides . Pesticides that are
not organic can stop the life processes of microbes, living things, plants and animals but some
of them can survive because of genetic changes or differences . Modern farming uses more
than four million tons of pesticides every year , and this makes some microbes more resistant
to pesticides. Also, some of these pesticides are man-made chemicals that have been around
for 50 years with the microbes in the environment; because they can be broken down by nature,
they can also be food for some microbes . When choosing pesticides for crops, it is important
to think about how harmful they are to the diseases that affect plants and the animals that live
on farms (they should be not harmful or very little) . Some researches have shown that using
certain or many kinds of pesticides on farms can make the pesticides build up in the soil , which
can have different effects on the microbes in the soil, animals and humans.The problem of
pesticide resistance is a serious threat to global health. Many studies have shown that bacteria
that can resist pesticides are found in various crops around the world. Bacteria in the soil can
quickly adjust to new situations, and the use or creation of new antimicrobial substances has
also raised the level of resistance among them . Pesticide resistance makes the soil bacteria
unaffected by pesticide applications, which allows them to live well . The main reason for the
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appearance and persistence of pesticide-resistant bacteria is the excessive use of pesticides .
Many bacteria that live in polluted environments have multiple resistance traits .The enzymatic
metabolism of pesticides can be mediated through oxidation, reduction, hydrolysis,
peroxidation and dehalogenation mechanisms . When ingested, inhaled, or absorbed dermally,
chlorpyrifos can be metabolized by cytochrome P450 enzymes, which can cause chlorpyrifos
to undergo dearylation (oxidative ester cleavage), forming 3,5,6-trichloro-2-pyridinol (TCP)
and diethylthiophosphate . Chlorpyrifos degradation mainly leads to 3,5,6-trichloro-2-
pyridinol (TCP), which is then degraded through bacterial enzymatic oxidation and hydrolytic
reactions. The TCP mechanism of degradation was demonstrated by Li et al., in 2010, who
suggested that TCP is broken down via the release of three chlorine atoms during its sequential
dechlorination in which one oxidative and two hydrolytic steps form 3,6-dihydroxypyridine-
2,5-dione . In another study, the degradation of 2,4-dichlorophenoxy acetic acid was shown to
have two different degradation pathways. In one pathway, the 6th carbon is oxidized through
the addition of an OH group, yielding 6-OH-2,4-D, and this reaction is followed by the removal
of acetate, resulting in the formation of 3,5-dichlorocatechol. In the other pathway, 2 carbon
side chains are removed, resulting in glyoxylate and 2,4-DCP. These two degradation pathways
were mediated by Pseudomonas sp., and Alcaligenes sp. isolates, respectively . The
oxygenases synthesized by Pseudomonas sp. have the ability to degrade tetrachlorobenzene to
trichlorocatechol by eliminating HCl from the compound . Mono and dioxygenases are actively
involved in the dehalogenation-mediated degradation of halogen-based pesticides (Rangasamy
et al., 2018). Proteomics has emerged as an excellent approach for gaining insight of
physiological changes at cellular level. Using proteome-based methods, we can directly
connect the breakdown of pesticides in nature to the proteins involved. Proteins are better
indicators than other biomolecules, because they show the real functions of metabolic and
regulatory processes and reveal more about the microbes’ activity than the genes or the
messenger RNA that code for them.(Pankaj et al., 2016)

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 ❖ REVIEW OF LITERATURE :

From the soil polluted by pesticides in the farm area of the University’s crop research centre,
they isolated a bacterium (SG4) that can break down cypermethrin. They identified it as
Bacillus thuringiensis strain SG4. It could degrade 78.9 % of cypermethrin (50 ppm) in 15 days
when we cultured it in a simple medium. They did a comparative proteomic analysis to find
out the proteins involved in cypermethrin degradation in this strain. They incubated it in a
simple medium with or without cypermethrin for 5 days and used 2D electrophoresis to detect
the proteins. They observed 223 and 250 different proteins in normal and stressed conditions
(cypermethrin exposure), respectively. We classified the proteins into different groups based
on their roles, such as catabolic enzymes, translational and stress proteins, etc. Studying the
proteins specific to cypermethrin degradation in this strain will help in improving the
biodegradation methods in the field.(Pankaj et al., 2016) . This is another research aimed to
discover the enzymes and metabolites that break down ß Cypermethrin, a pesticide that pollutes
the soil due to its frequent use in agriculture. The study used Bacillus cereus, a bacterium from
the soil of BT cotton crops that were exposed to pesticides, and found that it could degrade ß
Cypermethrin, an artificial pyrethroid insecticide that is widely used in farming. The
bioremediation process followed the standard lab protocol on mineral salt agar medium (MSM)
with ß Cypermethrin. The first step was to verify the breakdown of ß Cypermethrin by Thin-
Layer Chromatography (TLC), which showed the presence of metabolites with Retention
Factor (RF) values between 0.47 and 0.71, matching the standard samples of 3-PBA and
phenol. The next step was to identify the enzymes involved in the biodegradation by SDS-
PAGE analysis, which detected a protein of 56 kDa molecular weight and confirmed the
existence of Pyrethroid hydrolase. The final step was to validate the degradation of ß
Cypermethrin by GC–MS analysis, which recognized the metabolites as Benzylamine and
related compounds, which are derived from ß cypermethrin when degraded by B. cereus. The
study concluded that the 56 kDa molecular weight Pyrethroid hydrolase produced by B. cereus
could effectively metabolize the ß Cypermethrin pesticide. Therefore, B. cereus could be a
suitable candidate for chemical pesticide degradation.(Narayanan et al., 2020). The presence
of xenobiotics in their surroundings affects bacteria, who can either cope with or get rid of high
levels of them by breaking them down. A previous study reported a fast and effective
chlorpyrifos (CP) decomposer, Pseudomonas nitroreducens AR-3, and the current work aimed
to reveal how the bacterium does this. The bacterial reaction to CP involved changing the
proteome and activating global response pathways. Sixty-four out of the 794 significantly

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increased proteins were mainly involved in carbohydrate metabolism, environmental and
genetic information processing, synthesis of secondary metabolites and metabolism in various
environments. There was an increase of (mostly) periplasmic proteins, and those involved in
chemical breakdown pathways. Surprisingly, a novel reduction of stress response pathways
and xenobiotic transporter elements were also seen, possibly because CP was not seen as a
stress factor. A potential MBL-fold metallo-hydrolase protein with 44% sequence similarity to
an organophosphate hydrolase (ophC2) was found from the genome. Knowing the dynamic
changes in the protein profiles helped to understand the basic cellular and molecular
mechanisms behind CP decomposition and how the organism can survive under harsh
conditions.(Aswathi et al., 2021). Methyl parathion is a type of organophosphorus pesticide
that is used widely around the world to kill pests in farms and homes. But it is very harmful
and persistent in the environment, and it can damage the ecosystem and human health, leading
to many pollution incidents and human deaths every year. So it is very important to find and
study microorganisms that can completely break down methyl parathion and its breakdown
products, which can help clean up the sites that are polluted by pesticides. Burkholderia
zhejiangensis CEIB S4–3 is a kind of bacteria that was found in farm soils, and it can quickly
split methyl parathion at a level of 50 mg/L and get rid of all the p-nitrophenol that is released
in 12 hours when it grows in a simple salt solution. In this research, we compared the proteins
of this bacteria when it was exposed to methyl parathion or not, to see how it can break down
and resist methyl parathion. We looked at the changes in the proteins at three time points, zero,
three, and nine hours, using a technique called two-dimensional polyacrylamide gel
electrophoresis (2D-PAGE), and we identified the proteins that changed using another
technique called mass spectrometry (MALDI-TOF). We found 72 proteins that changed, 35
proteins when there was no pesticide, and 37 proteins when there was methyl parathion. These
proteins are involved in different ways of using and making energy, such as using sugars and
amino acids, making carbon compounds, burning fats, and breaking down aromatic
compounds, which include the enzymes that can break down p-nitrophenol in two ways
(Hydroquinone dioxygenase and Hydroxyquinol 1,2 dioxygenase). They also found that the
bacteria made more proteins that can protect it from cell damage, such as dealing with oxidative
stress, getting rid of harmful substances, and fixing DNA damage. These results show that B.
zhejiangensis CEIB S4–3 makes different proteins that can break down aromatic compounds
and p-nitrophenol, and it seems to prefer the Hydroquinone way of breaking down p-
nitrophenol, because it makes more of the enzyme Hydroquinone dioxygenase. The results also
show that the bacteria has ways of coping with the harmful effects of methyl parathion and p-
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nitrophenol.(Castrejón-Godínez et al., 2022). Chlorpyrifos is a widely used organophosphate
with many agricultural uses. It is a major source of soil pollution because of its high adsorption
coefficient, hydrophobicity and relatively long persistence. A study was conducted to observe
the reactions of Bacillus megaterium, which was isolated from a rice cultivation field, to
different levels of chlorpyrifos. The study showed that this bacterium could tolerate high
concentration (800 mg L-1) of chlorpyrifos. However, protein is a vital macromolecule in any
living cells and reflects all the essential functions happening inside the cell. Therefore,
measuring and profiling the protein of this bacterium in response to different levels of
chlorpyrifos would likely reveal expression, if any, of stress responsive gene(s). The overall
results indicated that Bacillus megaterium produced a few enzymes/ or proteins to adapt to the
environment. It can be a good degrader of chlorpyrifos, therefore, it could be used for cleaning
up chlorpyrifos polluted sites.(Shweta et al., 2023) . Imidacloprid is a common insecticide that
poses serious environmental threats. Its biodegradation can help estimate its remaining amount
in soil. Therefore, a soil microcosm-based study was conducted to evaluate the biodegradation
capacity of Agrobacterium sp. InxBP2. It achieved about 88% degradation in 20 days and
followed the pseudo-first-order kinetics (k = 0.0511 day−1 and t1/2=7 days). Whole genome
sequencing of Agrobacterium sp. InxBP2 showed a genome size of 5.44 Mbp with 5179 genes.
Imidacloprid degrading genes at loci K7A42_07110 (ABC transporter substrate-binding
protein), K7A42_07270 (amidohydrolase family protein), K7A42_07385 (ABC transporter
ATP-binding protein), K7A42_16,845 (nitronate monooxygenase family protein), and
K7A42_20,660 (FAD-dependent monooxygenase) that had sequence and functional similarity
with known counterparts were found. Molecular docking of proteins encoded by these genes
with their respective degradation pathway intermediates showed significant binding energies
(−6.56 to −4.14 kcal/mol). Molecular dynamic simulation revealed consistent interactions and
binding that indicated high stability of docked complexes. Proteome analysis showed
differential protein expression in imidacloprid treated versus untreated samples that supported
the in-silico results. Moreover, the detection of metabolites confirmed the bacterial degradation
of imidacloprid. Thus, the results established a mechanistic link between imidacloprid and
related degradative genes/enzymes of Agrobacterium sp. InxBP2. These findings will be very
important in performing the lifecycle analysis and developing strategies for the bioremediation
of soils polluted with insecticides like imidacloprid.(Gautam et al., 2023) .

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❖ RATIONALE :

Pesticide degrading bacteria are microorganisms that can break down pesticides into less
toxic or harmless compounds. Protein profiling and enzymatic study of these bacteria can
reveal the molecular mechanisms and pathways involved in pesticide degradation, as well
as the diversity and evolution of the degrading enzymes. .These studies can also help to
identify novel enzymes that can be used for bioremediation of pesticide contaminated sites,
or to engineer safer and more efficient pesticide degrading bacteria .

❖ AIM AND OBJECTIVES :

➢ To obtain and identify bacteria that can break down pesticide from contaminated
sites .
➢ To test the ability of bacteria to degrade pesticides in a simple medium .
➢ To detect the product of pesticide degradation by bacteria .
➢ To identify the enzyme involve and the expression of different protein in the
process of pesticide degradation .

❖ METHODOLOGY:

• Isolation and identification of pesticide degrading bacteria:

For isolation soil sample should be collected from contaminated site. Then the
bacteria will be isolated from this contaminated soil. Then the isolated bacteria
will be grown in appropriate medium with different concentration of pesticide.
Then the pure culture of the bacteria will be done o agar plate. Identifying them
by morphological and molecular methods, such as Gram staining, PCR
amplification.

• Protein extraction and quantification

Lysing the bacterial cells and extracting the total protein content using various
physical and chemical method .

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 • Protein profiling by SDS-PAGE

Solubilized proteins will be subjected to SDS-PAGE in gel slab. Electrophoresis

will be performed in a discontinuous buffer system, then the gel will be stained
by Coomassie brilliant blue, then the profile of the protein will be analysed.

• Screening of industrially important enzymes

There are different methods and techniques for screening of enzymes depending
on the types and source of the enzymes. Some techniques are like Crowded plate
technique, Indicator dye technique, Enrichment culture etc.

• Characterization of enzymes

Characterization of enzymes after screening is the process of determining the

biochemical and biophysical properties of the enzymes. There are different
methods for the characterization of the enzymes likes spectrophotometric assay,
electrophoretic assay, biochemical assay etc.

❖ SIGNIFICANCE IN PUBLIC HEALTH

➢ Understand the molecular mechanisms and genetic diversity of these bacteria , which
can enhance their bioremediation potential and efficiency .
➢ Identify and characterize the enzymes which are involved in pesticide degradation,
which can be used as biocatalysts for detoxifying pesticide residues in the environment
.
➢ Develop bioindicators for monitoring pesticide contamination and biodegradation in
soil and water .

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remodelling of Pseudomonas nitroreducens AR-3 potentially aid efficient degradation of the
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Martínez-Batallar ÁG, Hernández-Ortiz M, et al. Proteomic analysis of Burkholderia
zhejiangensis CEIB S4–3 during the methyl parathion degradation process. Pesticide
Biochemistry and Physiology 2022;187:105197.
https://doi.org/10.1016/j.pestbp.2022.105197.
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https://doi.org/10.1016/j.envres.2023.115271.
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Enzyme and metabolites attained in degradation of chemical pesticides ß Cypermethrin by
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https://doi.org/10.1016/j.matpr.2020.05.722.
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Pesticide degrading natural multidrug resistance bacterial flora. Microbial Pathogenesis
2018;114:304–10. https://doi.org/10.1016/j.micpath.2017.12.013.
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Bacillus spp 2023;36:1–11. https://doi.org/10.52228/JRUB.2023-36-1-1.